LOCUS       sequence-431513-        2993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) guide expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2993)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 2993)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA303
FEATURES             Location/Qualifiers
     source          1..2993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        444..692
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     444..464
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     615..634
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(903..919)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(903..919)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(916..938)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    927..943
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(951..957,958..975,976..981))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    996..1017
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1134..1151)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1305..1893)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1385..1404)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2071..2880)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2794..2813
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2872..2891
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagacgga agcgagggcc tatttcccat gattccttca tatttgcata
      481 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
      541 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      601 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      661 ctttatatat cttgtggaaa ggacgaaaca ccggagacgc ttttttcgtc tcagttgtag
      721 ctcccgaaac gttgctacaa taaggccgtc tgaaaagatg tgccgcaacg ctctgcccct
      781 taaagcttct gctttaaggg gcatcgttta tttttttgtt taaaccgtcc agtcttcgga
      841 accaaatcgg atcccgggcc cgtcgactgc agaggcctgc atgcaagctt ggtgtaatca
      901 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
      961 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1021 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1081 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     1141 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1201 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     1261 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     1321 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     1381 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     1441 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     1501 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     1561 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     1621 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     1681 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     1741 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     1801 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     1861 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     1921 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     1981 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaag cccaatctga
     2041 ataatgttac aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa
     2101 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     2161 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     2221 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     2281 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     2341 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     2401 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     2461 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     2521 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgtttttcc
     2581 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     2641 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     2701 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     2761 gcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     2821 atcagcatcc atgttggaat ttaatcgcgg cctcgacgtt tcccgttgaa tatggctcat
     2881 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt
     2941 tttatcttgt gcaatgtaac atcagagatt ttgagacacg ggccagagct gca
//
